ADP-ribosylation factors (ARFs) are a family of 20-kDa guanine nucleotide-binding proteins originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin. ARFs are highly conserved proteins found in all eukaryotic cells. At least six mammalian ARFs genes have been cloned; these can be grouped into three classes based on protein size, deduced amino acid sequence, phylogenetic analysis, and gene structure. Recombinant ARFs from all three classes activate cholera toxin-catalyzed ADP-ribosylation. Cytosolic or purified ARFs in the presence of GTPrS, associated with phospholipid or cell membranes. It has been reported that the amino terminus of ARF is a region critical for its activity in vitro and probably in vivo. A peptide with the amino acid sequence of the amino terminus of ARF1 reportedly inhibited ARF function, including its ability to enhance endosome fusion and Golgi transport as well as to activate cholera toxin. In this study, recombinant ARF1 (rARF1), rdelta13ARF1 (recombinant ARF1 lacking the first 13 amino acid) and rPKA14ARF1 (rARF1 in which the first 14 amino acids were replaced by the first seven amino acids of the cAMP-dependent protein kinase catalytic subunit) were used to assess the effect of the amino terminus on the ability of ARF to enhance ADP-ribosylation of agmatine by the cholera toxin A subunit. The GTP-dependent ARF activities of rdelta13ARF1 and rPKA14ARF1 were similar to that ARF1, whereas the GTP requirement for half-maximal activation of cholera toxin A, was somewhat higher for rARF1 than it was for rdelta13ARF1 and rPKA14ARF1. These results are consistent with the view that the amino terminus of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin.